Peptides and compositions which modulate apoptosis

ABSTRACT

The present invention is directed to novel peptides and compositions capable of modulating apoptosis in cells, and to methods of modulating apoptosis employing the novel peptides and compositions of the invention. In one aspect, the invention is directed to a novel peptide designated the &#34;GD domain,&#34; which is essential both to Bak&#39;s interaction with Bcl-x L , and to Bak&#39;s cell killing function. Methods of identifying agonists or antagonists of GD domain function are provided. The GD domain is responsible for mediating key protein/protein interactions of significance to the actions of multiple cell death regulatory molecules.

FIELD OF THE INVENTION

The present invention relates generally to the field of cell physiology,and more particularly, to programmed cell death, or apoptosis. The novelpeptides and compositions of the invention are useful for modulatingapoptosis in cells.

BACKGROUND OF THE INVENTION

The phenomenon of programmed cell death, or "apoptosis," is known to beinvolved in and important to the normal course of a wide variety ofdevelopmental processes, including immune and nervous system maturation.Apoptosis also plays a role in adult tissues having high cell turnoverrates (Ellis, R. E., et al., Annu. Rev. Cell. Biol. 7: 663-698 (1991);Oppenheim, R. W., Annu. Rev. Neurosci. 14: 453-501 (1991); Cohen, J. J.,et al. Annu. Rev. Immunol. 10: 267-293 (1992); Raff, M. C., Nature 356:397-400 (1992)). A number of different physiological signals normallyactivate programmed cell death in these contexts, but non-physiologicalinsults, such as irradiation and exposure to drugs which damage DNA,also can trigger apoptosis (Eastman, A., Cancer Cells 2: 275-280 (1990);Dive, C., et al., Br. J. Cancer 64: 192-196 (1991); Lennon, S. V., etal., Cell Prolif. 24: 203-214 (1991)).

In addition to its role in development, apoptosis has been implicated asan important cellular safeguard against tumorigenesis (Williams, G. T.,Cell 65: 1097-1098 (1991); Lane, D. P., Nature 362: 786-787 (1993)).Under certain conditions, cells die by apoptosis in response tohigh-level or deregulated expression of oncogenes (Askew, D., et al.,Oncogene 6: 1915-1922 (1991); Evan, G. I., et al., Cell 69: 119-128(1992); Rao, L., et al., Proc. Natl. Acad. Sci. USA 89: 7742-7746(1992); Smeyne, R. J., et al., Nature 363: 166-169 (1993); Tanaka, S.,et al., Cell 77: 829-839 (1994); Wu, X., et al., Proc. Natl. Acad. Sci.USA 91: 3602-3606 (1994)). Suppression of the apoptotic program, by avariety of genetic lesions, may contribute to the development andprogression of malignancies. This is well illustrated by the frequentmutation of the p53 tumor suppressor gene in human tumors (Levine, A.J., et al., Nature 351: 453-456 (1991)). Wild-type p53 is required forefficient induction of apoptosis following DNA damage (Clarke, A. R., etal., Nature 362: 849-852 (1993); Lowe, S. W., et al., Cell 74: 957-967(1993); Lowe, S. W., et al., Nature 362: 847-849 (1993)) and cell deathinduced by constitutive expression of certain oncogenes (Debbas, M., etal., Genes & Dev. 7: 546-554 (1993); Hermeking, H., et al., Science 265:2091-2093 (1994); Tanaka, S., et al., Cell 77: 829-839 (1994); Wu, X.,et al., Natl. Acad. Sci. USA 91: 3602-3606 (1994)). The cytotoxicity ofmany commonly used chemotherapeutic agents is mediated by wild-type p53(Lowe, S. W., et al., Cell 74: 957-967 (1993); Fisher, D. E., Cell 78:539-542 (1994)). Thus, loss of p53 function may contribute to theclinically significant problem of drug resistant tumor cells emergingfollowing chemotherapy regimens.

The expression product of the bcl-2 oncogene functions as a potentsuppressor of apoptotic cell death (McDonnell, T. J., et al., Cell 57:79-88 (1989); Hockenbery, D., et al., Nature 348: 334-336 (1990)).Constitutive Bcl-2 expression can suppress apoptosis triggered bydiverse stimuli, including growth factor withdrawal, oncogeneexpression, DNA damage, and oxidative stress (Vaux, D. L., et al.,Nature 335: 440-442 (1988); Sentman, C. L., et al., Cell 67: 879-888(1991); Strasser, A., et al., Cell 67: 889-899 (1991); Fanidi, A., etal., Nature 359: 554-556 (1992); Hockenbery, D. M., et al., Cell 75:241-251 (1993)). There is also conservation of Bcl-2 function acrossspecies. For example, the ced-9 gene of the nematode C. elegans appearsto be a structural and functional homolog of bcl-2 (Hengartner, M. O.,et al., Cell 76: 665-676 (1994)) and bcl-2 can complement ced-9mutations in transgenic animals (Vaux, D. L., et al., Science 258:1955-1957 (1991)). These observations suggest that Bcl-2 is intimatelyconnected with an evolutionarily conserved cell death program.

It is known that bcl-2 is a member of a family of related genes, atleast some of which also modulate apoptosis. Of these, bcl-x bears thehighest degree of homology to bcl-2, and is differentially spliced toproduce a long form, termed bcl-x_(L), and a shorter form, bcl-x_(S),related genes, at least some of which also modulate harboring aninternal deletion (Boise, L. H., et al., Cell 74: 597-608 (1993)).Bcl-x_(L) functions to suppress apoptosis, whereas the deleted form,Bcl-x_(S), inhibits the protection against cell death provided by Bcl-2expression. A second Bcl-2 homolog, Bax, forms heterodimers with Bcl-2(Oltvai, Z. N., et al., Cell 74: 609-619 (1993)) and has been shown tocounteract Bcl-2 and accelerate apoptosis. Mutational analysis of Bcl-2has suggested that the interaction with Bax is required for Bcl-2 tofunction as an inhibitor of cell death (Yin, X. -M., et al., Nature 369:321-323 (1994)).

The isolation and characterization of a bci-2 related gene, termed bak,is described in co-pending U.S. application Ser. No. 08/321,071, filed11 Oct. 1994, which is a continuation-in-part of U.S. application Ser.No. 08/287,427, filed 9 Aug. 1994, now abandoned (bak is referred totherein as bcl-y), the disclosures of which are incorporated herein byreference. Ectopic Bak expression accelerates the death of an IL-3dependent cell line upon cytokine withdrawal, and opposes the protectionagainst apoptosis afforded by Bcl-2. In addition, enforced expression ofBak is sufficient to induce apoptosis of serum deprived fibroblasts,raising the possibility that Bak directly activates, or is itself acomponent of, the cell death machinery.

The known cellular Bcl-2 related genes, where analyzed, have distinctpatterns of expression and thus may function in different tissues. WhileBcl-2 expression appears to be required for maintenance of the matureimmune system, it is desirable to identify other genes which may governapoptotic cell death in other lineages. In addition, the identificationof particular regions or domains of the proteins encoded by such genesmay provide a basis for understanding their structural and functionalcharacteristics and allow the development of valuable diagnostics andtherapeutics. For example, the identification of agents capable ofrestoring or inducing apoptosis in tumor cells (in which loss of p53tumor suppressor gene function may be implicated in tumorigenesis and inclinically significant drug resistance) would be of significanttherapeutic value, particularly where such restoration or induction wasindependent of p53 function. Similarly, the development of agentscapable of counteracting the anti-apoptotic function of oncogenes suchas as bcl-2, the activation of which is implicated in tumorigenesis(e.g., lymphoma) and in chemotherapeutic drug resistance, would be ofgreat potential value.

SUMMARY OF THE INVENTION

The present invention is directed to a novel protein domain of generalsignificance to the actions of multiple cell death regulatory molecules,which has been identified and mapped to a short subsequence in thecentral portion of the Bak molecule. This heretofore unrecognizedprotein domain, which the inventor has designated the "GD domain," isessential both to Bak'sinteraction with Bcl-x_(L), and to Bak's cellkilling function. Truncated Bak species encompassing the GD domain arethemselves sufficient to bind to Bcl-x_(L) and to kill cells intransfection assays.

The GD domain has been identified in two other Bcl-2 binding proteinsthat function to induce apoptosis: Bax and Bip1a. As with Bak, mutationof the homologous GD domain elements in Bax and Bip1a diminishes cellkilling and protein binding function. Thus, the GD domain is responsiblefor mediating key protein/protein interactions of significance to theactions of multiple cell death regulatory molecules.

In one aspect, then, the invention is directed to purified and isolatedpeptides comprising the GD domain and to molecules that mimic itsstructure and/or function, useful for inducing or modulating theapoptotic state of a cell. Chemical compounds that disrupt the functionof the GD domain have utility as apoptosis-modulating agents.Accordingly, in another aspect, the invention is directed to agentscapable of disrupting GD domain function. Such agents include, but arenot limited to, molecules that bind to the GD domain, molecules thatinterfere with the interaction of the GD domain with other protein(s),and molecules comprising the GD domain which is altered in some manner.The invention provides methods to identify molecules that modulateapoptosis by disrupting the function of the GD domain, which accordinglycomprise additional contemplated embodiments.

In additional aspects, the present invention relates to products andprocesses involved in the cloning, preparation and expression ofpeptides comprising the GD domain; antibodies with specificity to the GDdomain; and nucleotide sequences encoding the GD domain or portionsthereof. Peptides comprising the GD domain are useful for producingantibodies thereto. Such antibodies are useful for detecting andisolating proteins comprising the GD domain in biological specimensincluding, for example, cells from all human tissues including hearttissue, lung tissue, tumor cells, brain tissue, placenta, liver,skeletal muscle, kidney, and pancreas, as well as for modulating theapoptotic activity of proteins comprising the GD domain in and from suchbiological specimens, and constitute additional aspects of theinvention.

In yet another aspect, the invention provides for expression vectorscontaining genetic sequences, hosts transformed with such expressionvectors, and methods for producing the recombinant GD domain peptides ofthe invention.

The present invention is further directed to methods for inducing orsuppressing apoptosis in the cells and/or tissues of individualssuffering from degenerative disorders characterized by inappropriatecell proliferation or inappropriate cell death, respectively.Degenerative disorders characterized by inappropriate cell proliferationinclude, for example, inflammatory conditions, cancer, includinglymphomas, such as prostate hyperplasia, genotypic tumors, etc.Degenerative disorders characterized by inappropriate cell deathinclude, for example, autoimmune diseases, acquired immunodeficiencydisease (AIDS), cell death due to radiation therapy or chemotherapy,neurodegenerative diseases, such as Alzheimer's disease and Parkinson'sdisease, etc.

The present invention also relates to methods for detecting the presenceof the GD domain peptide, as well as methods directed to the diagnosisof degenerative disorders, which disorders are associated with anincreased or decreased level of expression of proteins comprising the GDdomain, as compared to the expected level of expression of such proteinsin the normal cell population.

The present invention relates to the therapeutic use of peptidescomprising the GD domain.

The present invention also relates to methods for modulating theapoptotic state of a cell by administering peptides comprising the GDdomain peptide, or mutants thereof, to an individual suffering from adegenerative disorder characterized by inappropriate cell proliferationor inappropriate cell death, in order to stabilize inappropriate cellproliferation (i.e., induce apoptosis) or stabilize inappropriate celldeath (i.e., suppress apoptosis), respectively, and/or in either case torestore normal cell behavior.

In another aspect, the present invention is related to the surprisingdiscovery that the Bak GD domain is involved in and sufficient forhomodimerization and heterodimerization of Bak. Nonlimiting examples ofBak GD domain dimerization include Bak (homodimerization), Bax(heterodimerization with a different killer protein) and Bcl-x_(L)(heterodimerization with a survival protein). Further, it hasunexpectedly been discovered that the non-essential regions of the Bakprotein in this aspect include the two domains in the carboxyl terminalhalf of the protein that show the highest degree of homology to otherBcl-2 family members (Bcl-2 homology domains I and II). Thus, peptidescomprising the GD domain are capable of mediating interactions not onlywith Bcl-x_(L), but also with Bak and Bax.

These and other objects and aspects of the invention will be apparent tothose of skill from the description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Cell killing function of Bak in different cell lines.

The indicated cell lines were co-transfected with a β-galactosidasemarker plasmid in combination with either a control plasmid (vector), ora plasmid expressing HA-epitope tagged Bak (HA-Bak). Cells were fixedand stained with X-gal at 24 hours post-transfection, and the number ofblue cells (β-galactosidase-positive) counted by microscopicexamination.

FIG. 2. Summary of the cell killing activity of Bak deletion mutants andtruncated species.

The structures of the various Bak mutants are illustrated schematically.The precise amino acid region(s) removed by deletion are indicated bythe numbers at the left (SEQ ID NOS:11 and 1). The endpoints of the Bakamino acid residues retained in the truncated species (bottom, QVG andPEM) are indicated by numbers bordering the schematics of theirrespective structures. The Rat-1 cell killing activity is summarized asfollows: +, cell killing capacity equivalent to wild-type Bak; -, nocell killing activity; +/-, diminished cell killing activity relative towild-type Bak. nd indicates experiment not done.

FIGS. 3A-3B. Interaction of Bak with Bcl-x_(L).

A). Bak/Bcl-x_(L) interactions measured in vitro. ³⁵ S labeled in vitrotranslated Bak (lane 1) was mixed either with GST-BCl-x_(L) (lane 2) orGST (lane 3). The complexes were captured on glutathione-agarose beads,and bound ³⁵ S labeled Bak protein was detectedby electrophoresis on SDSpolyacrylamide gels followed by autoradiography.

B). Bak/Bcl-x_(L) interactions detected in transfected cells. Plasmidsexpressing epitope-tagged forms of Bak and Bcl-x_(L) (HA-Bak and Flagtag-Bcl-x_(L)) were co-transfected into COS cells. HA-Bak wasimmunoprecipitated (anti-HA IP) from transfected cell lysates andassociated Bcl-x_(L) was detected by Western blot analysis with ananti-Flag tag antibody.

FIG. 4. Summary of Bcl-x_(L) binding function of Bak deletions andtruncated species.

The structures of the various Bak mutants are shown schematically, asdescribed in FIG. 2 (SEQ ID NOS:11 and 1). The capacity of the Bakmutants and truncated species to interact with Bcl-x_(L) is summarized(right) as follows: +, equivalent to wild type Bak in ability tointeract with Bcl-x_(L) both in vitro and in transfected COS cells; -,no interaction with Bcl-x_(L) detected; -/+, interaction greatlydiminished relative to wild-type Bak and could only be detected invitro.

FIG. 5. Regions homologous to the Bak GD domain are present in Bip1a andBax.

Top. Schematic structures of the proteins with the positions of the GDdomain homology (open boxes), hydrophobic segment (hatched boxes) andBcl-2 homology domains (filled boxes).

Bottom. Amino acid sequence of the regions in Bip1a (SEQ ID NO:12) andBax (SEQ ID NO:13) homologous to the Bak GD domain (SEQ ID NO: 14).Highlighted residues are identical in at least two of the proteins;shaded residues indicate conservative amino acid changes. Also shown(solid lines) are the amino acid regions removed in the indicated Bip1a,Bak and Bax deletion mutants.

FIG. 6. Summary of cell killing and Bcl-x_(L) binding activities of GDdomain deletion mutants.

The data for cell killing and Bcl-x_(L) binding function are summarizedas described in FIG. 2 and FIG. 4, respectively.

FIG. 7. Bak GD domain dimerization.

Interactions of the Bak GD domain with Bak and Bax were measuredessentially as described for Bak binding to Bcl-x_(L). A portion of Bak(PEM) encompassing the GD domain (residues 58-103) was fused to GST, tocreate GST-PEM. In vitro translated, ³⁵ S labeled Bcl-x_(L), Bak, Baxand Bip1a were incubated with either GST alone, or GST-PEMbacterially-expressed fusion proteins. Complexes were captured withglutathione-agarose beads, washed, and bound proteins detected bypolyacrylamide gel electrophoresis and autoradiography. Bcl-x_(L), Bak,and Bax all interact specifically with GST-PEM, but not GST alone. Thus,the GD domain can mediate interaction not only with Bcl-x_(L) but alsoBak and Bax.

FIGS. 8A-8C. DNA sequence encoding the GD domain in Bak, Bax and Bip1a.

DNA sequences encoding the GD domain regions for Bak (A-D, SEQ IDNOS:15, 17, 19 and 21), Bax (E-G, SEQ ID NOS:23, 25 and 27) and Bip1a(H-J, SEQ ID NOS:29, 31 and 33) are shown along with their correspondingamino acid sequences (SEQ ID NOS:16, 18, 20, 22, 24, 26, 28, 30, 32 and34). The nucleotide numbers above each sequence are based on starting atthe initiating ATG for each protein. The underlined number refers to theposition of the first and last amino acid of the peptide shown.

DETAILED DESCRIPTION OF THE INVENTION

Technical and scientific terms used herein have the meanings commonlyunderstood by one of ordinary skill in the art to which the presentinvention pertains, unless otherwise defined. Reference is made hereinto various methodologies known to those of skill in the art.Publications and other materials setting forth such known methodologiesto which reference is made are incorporated herein by reference in theirentireties as though set forth in full. Standard reference works settingforth the general principles of recombinant DNA technology includeSambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory Press, Planview, N.Y. (1989); McPherson,M. J., Ed., Directed Mutagenesis: A Practical Approach, IRL Press,Oxford (1991); Jones, J., Amino Acid and Peptide Synthesis, OxfordScience Publications, Oxford (1992); Austen, B. M. and Westwood, O. M.R., Protein Targeting and Secretion, IRL Press, Oxford (1991). Anysuitable materials and/or methods known to those of skill can beutilized in carrying out the present invention; however, preferredmaterials and/or methods are described. Materials, reagents and the liketo which reference is made in the following description and examples areobtainable from commercial sources, unless otherwise noted.

A previously unrecognized domain within the Bak molecule that appears tobe both necessary and sufficient for the known biological activities ofBak has now been identified. This domain, designated herein as the "GDdomain," is sufficient to mediate cell killing function and physicalinteraction with Bcl-xL. Sequences homologous to the Bak GD domain havealso been identified within Bax and Bip1a and shown to be similarlyrequired for the cell killing and Bcl-x_(L) binding activities of theseproteins. These observations suggest that Bak, Bax and Bip1a modulate orregulate apoptosis through a similar mechanism that, in each case,involves their respective GD domains. As those of skill familiar withthe present invention will appreciate, sequences comprising the GDdomain are useful in modulating apoptosis in cells. Similarly, compoundsand compositions which are capable of binding to the GD domain areuseful as agents for the modulation of apoptotic activity in cells.

As used herein, the term "GD domain" refers to a protein domain firstidentified in Bak, demonstrated herein to be essential for theinteraction of Bak with Bcl-x_(L) and for Bak's cell killing function,and to peptides and/or molecules capable of mimicking its structureand/or function. In a preferred embodiment, the present inventioncomprises a peptide having the following amino acid sequence:

    GDDINRRYDSEFQ [SEQ ID NO:1]

corresponding to amino acid residues 82-94 of Bak, as well as functionalequivalents thereof. By "functional equivalent" is meant a peptidepossessing a biological activity or immunological characteristicsubstantially similar to that of the GD domain, and is intended toinclude "fragments", "variants", "analogs", "homologs", or "chemicalderivatives" possessing such activity or characteristic. Functionalequivalents of the GD domain, then, may not share an identical aminoacid sequence, and conservative or non-conservative amino acidsubstitutions of conventional or unconventional amino acids arepossible.

Reference herein to "conservative" amino acid substitution is intendedto mean the interchangeability of amino acid residues having similarside chains. For example, glycine, alanine, valine, leucine andisoleucine make up a group of amino acids having aliphatic side chains;serine and threonine are amino acids having aliphatic-hydroxyl sidechains; asparagine and glutamine are amino acids having amide-containingside chains; phenylalanine, tyrosine and tryptophan are amino acidshaving aromatic side chains; lysine, arginine and histidine are aminoacids having basic side chains; and cysteine and methionine are aminoacids having sulfur-containing side chains. Interchanging one amino acidfrom a given group with another amino acid from that same group would beconsidered a conservative substitution. Preferred conservativesubstitution groups include asparagine-glutamine, alanine-valine,lysine-arginine, phenylalanine-tyrosine and valine-leucine-isoleucine.

In a preferred embodiment of the invention, there is provided a peptidehaving the following amino acid sequence:

    PSSTMGQVGRQLAIIGDDINRRYDSEFQ [SEQ ID NO:2]

corresponding to amino acid residues 67-94 of Bak, uniquely required forBak cell killing function.

In another preferred embodiment, there is provided a peptide having thefollowing amino acid sequence:

    QVGRQLAIIGDDINRRYDSEFQTMLQHLQPT [SEQ ID NO:3]

corresponding to amino acid residues 73-103 of Bak, sufficient for thecell killing function of Bak.

The present data indicate that the biological activity of the GD domainand its functional derivatives will be affected by the sub-cellularlocalization of these compositions. Accordingly, in another preferredembodiment of the invention, the GD domain peptides of the inventionwill have fused to their C-terminal end an appropriate hydrophobic tail,which may comprise amino acids 187-211 of Bak. Other suitable means ofeffecting sub-cellular localization, including the selection of suitablehydrophobic tails, such as amino acids 172-192 of Bax, amino acids213-233 of Bcl-x_(L), amino acids 220-240 of Bcl-2, and hydrophobictails introduced through protein lipidation (Casey, T. J., Science, 268:221-225 (1995)) such as prenylation and acylation (e.g., myristylation,palmitylation) may be employed by those of skill using known methods.

The GD domain disclosed herein is uniquely involved in both cell killingand Bcl-x_(L) binding activity of Bak. Moreover, other Bcl-2 interactingproteins having functional properties resembling those of Bak aredemonstrated herein to contain amino acid regions having sequencesbearing homology to sequences within the GD domain of Bak. Theseproteins include Bax and Bip1a which, like Bak, interact with Bcl-2, andboth of these proteins contain amino acid regions bearing homology tosequences within the GD domain of Bak. In Bax, this region comprisesamino acids 59-73, which bears homology to amino acids 74-88 within theGD domain of Bak. The protein Bip1a similarly contains an amino acidregion comprising amino acids 57-71 bearing homology to the samesequences (amino acids 74-88) within the Bak GD domain. Deletion of theBax and Bip1a GD domain regions identified above impaired their cellkilling activity and prevented binding to Bcl-x_(L). Bip1a lackssequences homologous to the two highly conserved regions, designatedDomain I and Domain II (also referred to in the literature as "Bcl-2Homology domains" or "BH domains" I and II or "BH1" and "BH2"). It hasbeen suggested that these two conserved regions, and especially DomainI, are instrumental in dictating homo- and heterodimerization in Bcl-2,Bax, and other Bcl-2 family members. Accordingly, the GD domainconstitutes a key element involved in the biological activity ofproteins such as Bak, Bax and Bip1a, not necessarily shared with Bcl-2family members, which activity is independent of BH domains I and II.This suggests that the GD domain defines a distinct family of proteins,including Bak, Bax and Bip1a.

Accordingly, in an additional preferred embodiment, there is provided apeptide comprising the following amino acids:ps ti LSECLKRIGDELDSN [SEQID NO:4]

corresponding to amino acids 59-73 of Bax. In another preferredembodiment, a peptide is provided which comprises amino acid sequence:

    LKRIGDELD [SEQ ID NO:5]

corresponding to amino acids 63-71 of Bax. In another preferredembodiment, a peptide is provided which comprises amino acid sequence:

    QDASTKKLSECLKRIGDELDSNMELQ [SEQ ID NO:6]

corresponding to amino acids 52-77 of Bax. In another preferredembodiment, a peptide is provided which comprises amino acid sequence:

    LALRLACIGDEMDVS [SEQ ID NO:7]

corresponding to amino acids 57-71 of Bip1a. In another preferredembodiment, there is provided a peptide comprising the following aminoacid sequence:

    IGDEM [SEQ ID NO:8]

corresponding to amino acids 64-68 of Bip1a. In another preferredembodiment, there is provided a peptide comprising the following aminoacid sequence:

    CMEGSDALALRLACIGDEMDVSLRAPRL [SEQ ID NO:9]

corresponding to amino acids 50-77 of Bip1a. In another preferredembodiment, there is provided a peptide comprising the following aminoacid sequence:

    VGRQLAIIGDDINRR [SEQ ID NO:10]

corresponding to amino acids 74-88 of Bak.

A surprising aspect of the present invention is the discovery that theGD domain alone is sufficient for homodimerization of Bak, as well asfor heterodimerization of Bak with Bax and Bcl-x_(L), and that thehighly conserved Bcl-2 family Domains I and II are not necessary forthis dimerization. This indicates that the GD domain is capable ofmodulating the function of proteins including Bak, Bax and Bcl-x_(L)directly through dimerization, and thus may also modulate the functionof other proteins including Bcl-2.

The functional importance of the GD domain, then, is likely to berelated to its ability to mediate one or more protein/proteininteractions with other Bcl-2 family members, or with other as yetunidentified cellular protein(s). It is possible that survival proteinslike Bcl-2 and Bcl-x_(L) suppress apoptosis by binding and inactivatingproteins that actively promote cell death, such as Bak, Bax and Bip1a,through their GD domains. In support of this view, the interaction withBax appears to be required for Bcl-2 to suppress apoptosis (Yin et al.,Nature 369: 321-323 (1994)). A second possibility is that Bak, Bax, andBip1a induce cell death bybinding (via their GD domains) andinactivating proteins, including Bcl-2 and Bcl-x_(L), that activelypromote cell survival. It is also possible that Bak, Bax and Bip1a bindone or more additional cellular proteins and that this interactionmediates cell death function. The present inventor does not intend to bebound by a particular theory; however, regardless of its mechanism(s) ofaction, the GD domain in Bak, Bax and Bip1a is of central importance formediating these protein/protein interactions.

Agents capable of modulating GD domain mediated protein/proteininteractions may include peptides comprising the GD domain, as well asmutants of the GD domain or of proteins comprising the GD domain. A"mutant" as used herein refers to a peptide having an amino acidsequence which differs from that of the naturally occurring peptide orprotein by at least one amino acid. Mutants may have the same biologicaland immunological activity as the naturally occurring GD domain peptideor the naturally occurring protein. However, the biological orimmunological activity of mutants may differ or be lacking. For example,a GD domain mutant may lack the biological activity which characterizesnaturally occurring GD domain peptide, but may be useful as an antigenfor raising antibodies against the GD domain or for the detection orpurification of antibodies against the GD domain, or as an agonist(competitive or non-competitive), antagonist, or partial agonist of thefunction of the naturally occurring GD domain peptide.

Modulation of GD domain mediated protein/protein interactions may beeffected by agonists or antagonists of GD domain peptides as well.Screening of peptide libraries, compound libraries and other informationbanks to identify agonists or antagonists of the function of proteinscomprising the GD domain is accomplished with assays for detecting theability of potential agonists or antagonists to inhibit or augment GDdomain binding, e.g., GD domain homodimerization or heterodimerization.

For example, high through-put screening assays may be used to identifycompounds that modulate the protein binding function of the GD domain.Such screening assays facilitate the identification of compounds thataccelerate or inhibit apoptosis by influencing protein/proteininteractions mediated by the GD domain. For example, an in vitro screenfor compounds that disrupt the Bak GD domain interaction withGST-Bcl-x_(L) comprises multiwell plates coated with GST-Bcl-x_(L) whichare incubated with a labeled GD domain peptide probe in the presence ofone or more compounds to be tested. Molecules that specifically disruptthe interaction could, in principle, bind to either the GD domain"ligand" or to the as yet undefined "receptor" domain in Bcl-x_(L).Either class of compound would be a candidate apoptosis-modulatingagent.

Thus, the invention provides a method of screening for an agent capableof modulating apoptosis which comprises coating a multiwell plate withGST-Bcl-x_(L) and incubating the coated multiwell plate with a labeledGD domain peptide probe in the presence of an agent which it is desiredto test, wherein disruption of GD domain interaction with GST-Bcl-x_(L)indicates that said agent is capable of modulating apoptosis. Agentsidentified by this method are also contemplated embodiments of theinvention.

Suitable labels include a detectable label such as an enzyme,radioactive isotope, fluorescent compound, chemiluminescent compound, orbioluminescent compound. Those of ordinary skill in the art will know ofother suitable labels or will be able to ascertain such using routineexperimentation. Furthermore, the binding of these labels to thepeptides is accomplished using standard techniques known in the art.

A high speed screen for agents that bind directly to the GD domain mayemploy immobilized or "tagged" combinatorial libraries. Agents that bindspecifically to such libraries are candidates to be tested for theircapacity to block Bak/Bcl-x_(L) interactions. As discussed above, suchagents may function as suppressors of apoptosis by either directlyinhibiting Bak (and/or Bax/Bip1a) function, or by increasing theeffective activity of endogenous Bcl-2/Bcl-x_(L) (or other Bcl-2 familymember). Such agents would be useful for suppressing aberrant apoptosisin degenerative disorders or following ischemic injury.

Antibodies against the GD domain peptides of the invention may be usedto screen cDNA expression libraries for identifying clones containingcDNA inserts encoding structurally related, immunocrossreactive proteinswhich may be members of the GD domain family of proteins. Screening ofcDNA and mRNA expression libraries is known in the art. Similarly,antibodies against GD domain peptides are used to identify or purifyimmunocrossreactive proteins related to this domain, or to detect ordetermine the amount of proteins containing the GD domain in a cell orcell population, for example, in tissue or cells, such as lymphocytes,obtained from a patient. Known methods for such measurements includeimmunoprecipitation of cell extracts followed by PAGE, in situ detectionby immunohistochemical methods, and ELISA methods, all of which are wellknown in the art.

Modulation of apoptosis according to the invention includes methodsemploying specific antisense polynucleotides complimentary to all orpart of the nucleotide sequences encoding proteins comprising the GDdomain disclosed herein. Such complimentary antisense polynucleotidesmay include nucleotide additions, deletions, substitutions andtranspositions, providing that specific hybridization to the targetsequence persists. Soluble antisense RNA or DNA oligonucleotides whichcan hybridize specifically to mRNA species encoding proteins comprisingthe GD domain, and which prevent transcription of the mRNA speciesand/or translation of the encoded polypeptide are contemplated ascomplimentary antisense polynucleotides according to the invention.Production of proteins comprising the GD domain is inhibited byantisense polynucleotides according to the invention, and such antisensepolynucleotides may inhibit apoptosis, senescence and the like, and/orreverse the transformed phenotype of cells. A heterologous expressioncassette maybe used to produce antisense polynucleotides in atransfectant or transgenic cell. Antisense polynucleotides also may beadministered as soluble oligonucleotides to the external environment ofthe target cell, such as the culture medium of cells in vitro or theinterstitial fluid (e.g., via the circulatory system) in vivo. Antisensepolynucleotides and their use are known to those of skill, and aredescribed, for example, in Melton, D. A., Ed, Antisense RNA and DNA,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988).

The predicted biological activity of agents identified according to theinvention varies depending on the assumptions made regarding themechanism of Bak/Bcl-2 function. For example, an agent which bindstightly to the GD domain would be predicted to inhibit Bak (and perhapsBax/Bip1a) function. Assuming Bak (and/or Bax/Bip1a) is the active celldeath regulatory molecule, an agent that binds tightly to the GD domainmay inhibit Bak function via a mechanism similar to the action ofBcl-2/Bcl-x_(L) binding. Such agents would comprise "Bcl-2/Bcl-x_(L) "mimetics and might, therefore, exhibit anti-apoptotic activity underconditions in which Bcl-2 has a demonstrated protective effect (e.g.,protection of neurons against injury or cytokine deprivation). Agents inthis class could have utility in treating diseases characterized byexcessive or inappropriate cell death, including, for example,neuro-degenerative diseases and injury resulting from ischemia.

If Bcl-2/Bcl-x_(L) binding actively promotes cell survival, and if Bakrepression is due simply to its binding and inactivating these survivalproteins, then an agent that prevented this binding would effectivelyincrease the activity of resident Bcl-2/Bcl-x_(L) in a cell by relievingrepression by Bak (and/orbyBax/Bip1a). This would also promote cellsurvival, but only in cells that express endogenous Bcl-2/Bcl-x_(L).Agents that bind to Bcl-x_(L) and thereby prevent its interaction withBak (and/or with Bax/ Bip1a) might inhibit the cell death suppressionactivity of Bcl-x_(L) (and/or of Bcl-2). Such agents would comprise "GDdomain mimetics" and would promote cell death in a fashionmechanistically similar to the action of Bak. GD domain mimetic agentswould be useful in the therapeutic treatment of cancer and viraldisease.

Peptidomimetics of GD domain peptide are also provided by the presentinvention, and can act as drugs for the modulation of apoptosis by, forexample, blocking the function of proteins comprising the GD domain orinterfering with GD domain mediated dimerization. Peptidomimetics arecommonly understood in the pharmaceutical industry to includenon-peptide drugs having properties analogous to those of those of themimicked peptide. The principles and practices of peptidomimetic designare known in the art and are described, for example, in Fauchere J.,Adv. Drug Res. 15: 29 (1986); and Evans et al., J. Med. Chem. 30: 1229(1987). Peptidomimetics which bear structural similarity totherapeutically useful peptides may be used to produce an equivalenttherapeutic or prophylactic effect. Typically, such peptidomimetics haveone or more peptide linkages optionally replaced bya linkage which mayconvert desirable properties such as resistance to chemical breakdown invivo. Such linkages may include --CH₂ NH--, --CH₂ S--, --CH₂ --CH₂ --,--CH═CH--, --COCH₂ --, --CH(OH)CH₂ --, and --CH₂ SO--. Peptidomimeticsmay exhibit enhanced pharmacological properties (biological half life,absorption rates, etc.), different specificity, increased stability,production economies, lessened antigenicity and the like which makestheir use as therapeutics particularly desirable.

As discussed herein, the GD domain appears to be an area of motifsinvolved in dimerization, and this activity may be related to theregulation of apoptosis by proteins comprising the GD domain. Bakpossesses a C-terminal hydrophobic region which appears to be membranespanning. Thus, sub-cellular localization of proteins containing the GDdomain may play a role in the regulation of programmed cell death invivo. It is possible, then, to employ the invention for detection ordetermination of proteins comprising the GD domain, for example, infractions from tissue/organ excisions, by means of immunochemical orother techniques in view of the antigenic properties thereof.Immunization of animals with peptides comprising the GD domain alone orin conjunction with adjuvants by known methods can produce antibodiesspecific for the GD domain peptide. Antiserum obtained by conventionalprocedures may be utilized for this purpose. For example, a mammal, suchas a rabbit, may be immunized with a peptide comprising the GD domain,thereby inducing the formation of polyclonal antibodies thereagainst.Monoclonal antibodies also may be generated using known procedures. Suchantibodies can be used according to the invention to detect the presenceand amount of peptides comprising the GD domain.

The GD domain peptides of the invention may be used for the detection ofBak, Bcl-x_(L), Bip1a and other proteins by means of standard assaysincluding radioimmunoassays and enzyme immunoassays.

It will be appreciated by those of skill that the precise chemicalstructure of peptides comprising the GD domain will vary depending upona number of factors. For example, a given protein may be obtained as anacidic or basic salt, or in neutral form, since ionizable carboxyl andamino groups are found in the molecule. For the purposes of theinvention, then, any form of the peptides comprising the GD domain whichretains the therapeutic or diagnostic activity of the naturallyoccurring peptide is intended to be within the scope of the presentinvention.

The GD domain peptides and other compositions of the present inventionmay be produced by recombinant DNA techniques known in the art. Forexample, nucleotide sequences encoding the GD domain peptides of theinvention may be inserted into a suitable DNA vector, such as a plasmid,and the vector used to transform a suitable host. The recombinant GDpeptide is produced in the host by expression. The transformed host maybe a prokaryotic or eukaryotic cell. Preferred nucleotide sequences forthis purpose encoding the GD domains of Bak, Bax and Bip1a are set forthin FIG. 8.

Polynucleotides encoding peptides comprising the GD domain may begenomic or cDNA, isolated from clone libraries by conventional methodsincluding hybridization screening methods. Alternatively, syntheticpolynucleotide sequences may be constructed by known chemical syntheticmethods for the synthesis of oligonucleotides. Such synthetic methodsare described, for example, in Blackburn, G. M. and Gait, M. J., Ed.,Nucleic Acids in Chemistry and Biology, IRL Press, Oxford, England(1990), and it will be evident that commercially availableoligonucleotide synthesizers also may be used according to themanufacturer's instructions. One such manufacturer is Applied BioSystems.

Polymerase chain reaction (PCR) using primers based on the nucleotidesequence data disclosed herein may be used to amplify DNA fragments frommRNA pools, cDNA clone libraries or genomic DNA. PCR nucleotideamplification methods are known in the art and are described, forexample, in Erlich, H. A., Ed., PCR Technology: Principles andApplications for DNA Amplification, Stockton Press, New York, N.Y.(1989); U.S. Pat. No. 4,683,202; U.S. Pat. No. 4,800,159; and U.S. Pat.No. 4,683,195. Various nucleotide deletions, additions and substitutionsmay be incorporated into the polynucleotides of the invention as will berecognized by those of skill, who will also recognize that variation inthe nucleotide sequence encoding GD domain peptides may occur as aresult of, for example, allelic polymorphisms, minor sequencing errors,and the like. The polynucleotides encoding GD domain peptides of theinvention may include short oligonucleotides which are useful, forexample, as hybridization probes and PCR primers. The polynucleotidesequences of the invention also may comprise a portion of a largerpolynucleotide and, through polynucleotide linkage, they may be fused,in frame, with one or more polynucleotide sequences encoding differentproteins. In this event, the expressed protein may comprise a fusionprotein. Of course, the polynucleotide sequences of the invention may beused in the PCR method to detect the presence of mRNA encoding GD domainpeptides in the diagnosis of disease or in forensic analysis.

cDNAs encoding proteins which interact with the GD domain (or proteinscontaining the GD domain) can be identified by screening cDNA expressionlibraries, employing known methods. Examples of such methods include theyeast two-hybrid system (U.S. Pat. No. 5,283,173, inventors Fields andSong, issued Feb. 1, 1994; Chien, et al., Proc. Natl. Acad. Sci. 88:9578 (1991), and the E. coli/BCCP interactive screening system(Guarente, L., Proc. Natl. Acad. Sci. 90: 1639 (1993) and Germino, etal., Proc. Natl. Acad. Sci. 90: 933-937 (1993)). Suitable cDNA librarieswill include mammalian cDNA libraries, such as human, mouse or rat,which may contain cDNA produced from RNA and a single cell, tissue ororgan type or developmental stage, as are know in the art.

A nucleotide sequence encoding a protein or peptide comprising the GDdomain may be inserted into a DNA vector in accordance with conventionaltechniques, including blunt-ending or staggered-ending termini forligation, restriction enzyme digestion to provide appropriate termini,filling in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and ligation with appropriateligases. Techniques for such manipulations are disclosed, for example,by Sarabrook, J., et al., Molecular Cloning: A Laboratory Manual, 2dEd., Cold Spring Harbor Laboratory Press, Planview, N.Y. (1989), and arewell known in the art.

The sequence of amino acid residues in a protein or peptide comprisingthe GD domain is designated herein either through the use of theircommonly employed three-letter designations or by their single-letterdesignations. A listing of these three-letter and one-letterdesignations may be found in textbooks such as Biochemistry, SecondEdition, Lehninger, A., Worth Publishers, New York, N.Y. (1975). Whenthe amino acid sequence is listed horizontally, the amino terminus isintended to be on the left end whereas the carboxy terminus is intendedto be at the right end. The residues of amino acids in a peptide may beseparated by hyphens. Such hyphens are intended solely to facilitate thepresentation of a sequence.

The rational design of GD domain mimetics or binding molecules, based onmodeled (or experimentally determined) peptide structure, may be carriedout by those of skill, using known methods of rational drug design.Therapeutic or prophylactic methods for treating pathological conditionssuch as autoimmune disease, neurodegenerative disease, cancer and thelike, are accomplished by the administration of an effective amount of atherapeutic agent capable of specifically inhibiting GD domainhomodimerization or heterodimerization, thereby modulating thebiological activity of GD domain containing proteins and the apoptoticstate in a patient.

Truncated Bak molecules comprising the GD domain, such as QVG or PEM, aswell as other small peptide derivatives that constitute a "minimal" GDdomain, are demonstrated herein to retain the protein binding and cellkilling function exhibited by wild-type Bak. These molecules, orpeptidomimetic derivatives, may induce apoptosis in tumor cells byproviding the same biological signal produced by high level expressionof Bak (which has been shown to kill tumor cells in an in vitro assay).Such agents comprise a novel class of chemotherapeutic drug that wouldbe predicted to operate independently of p53 status.

If interaction with Bak results in the suppression of the anti-apoptoticfunction of Bcl-x_(L) and/or other Bcl-2 family members, then GD domainpeptides, or agents that mimic the GD domain structure, may act asinhibitors of the anti-apoptotic function of proteins like Bcl-2. Highlevel Bcl-2 expression has been implicated in the resistance of tumorcells to a variety chemotherapy drugs (Fisher et al., Cancer Res. 53:3321-3326 (1993); Miyashita and Reed, Blood 81: 151-157 (1993); Dole etal., Cancer Res. 54: 3253-3259 (1994). Administration of GD domainmimetics may suppress Bcl-2 function and restore sensitivity of tumorcells to apoptosis induced by traditional chemotherapeutic agents. Inaddition, Bak or GD domain mimetics that inhibit Bcl-2 may themselves beselectively toxic to certain tumors, such as follicular lymphoma, thatdepend upon high level Bcl-2 activity for their continued growth andsurvival.

The GD domain mimetics of the invention may also have utility incombating vital infections. Apoptosis of infected cells, with associatedDNA fragmentation, provides an important defense against viralpathogenesis by limiting vital titers and restricting viral propagation(Vaux et al., Cell 76: 777-779 (1994). For this reason, viruses haveevolved diverse mechanisms to suppress apoptosis of infected host cells.Certain viral proteins, such as Epstein-Barr virus BHRF-1, African SwineFever Virus (ASFV) LHW5-HL, and Adenovirus E1B 19kD, appear to bestructural or functional homologues of Bcl-2. A second Epstein-Barrvirus gene, LMP1, transactivates the expression of the cellular bcl-2gene in latently infected cells (Henderson et al., Cell 65: 1107-1115(1991). In these cases, the apoptotic signal triggered by viralinfection may be held in check by the action of a vital (or cellular)Bcl-2 homolog. A Bak GD domain mimetic that opposes the anti-apoptoticfunction of the viral/cellular Bcl-2 homolog would serve to alleviatethis block and induce apoptosis in infected cells and consequentlyinhibit vital propagation. Anti-apoptotic proteins encoded by at leasttwo unrelated viruses (EBV BHRF1 and Adenovirus E1B 19kD) have beendemonstrated to interact with Bak. Experimental evidence supports theconclusion that disrupting the E1B 19kD/Bak interaction (i.e., bycompeting with a GD domain mimetic) would reduce vital titers andproductive replication. Mutations in E1B 19kD that disrupt theinteraction with Bak correspondingly abolish the anti-apoptotic functionof E1B 19kD. Adenovirus strains encoding defective E1B 19kD proteinsyield much lower progeny virus in vitro, due to apoptosis of infectedcells (Pilder et al., J. Virol. 52: 664-671 (1984); Subramanian et al.,J. Biol. Chem. 259: 11777-11783 (1984).

An additional mechanism whereby viruses impose a blockade on theapoptosis signal transduction pathway is through the inactivation of thep53 tumor suppressor protein. Forced cellular proliferation caused byviral infection induces an apoptotic signal that requires p53 function(see e.g., Wu and Levine, Proc. Natl. Acad. Sci. USA 91: 3602-3606(1994). Typically, p53 function is abrogated during infection byphysical interaction with a viral gene product. Examples of viruses thatencode p53 binding proteins include adenoviruses, polyoma viruses,papilloma viruses, and cytomegalovirus (Levine et al., Nature 351:453-456 (1991); Speir et al., Science 265: 391-394 (1994). Infectedcells are "primed" to undergo apoptosis, but cell death is prevented ordelayed by viral inhibition of p53 function. It is possible that thisblockade in the apoptosis signal transduction pathway could be relieved,or bypassed, by an agent that modulates apoptosis downstream of p53.Bak, or GD domain mimetics, induce apoptosis independently of p53, andconsequently provide a way to implement or restore the cell death signalthat is suppressed in infected cells.

Any mode of administration which results in the delivery of thetherapeutic agent across the cell membrane and into the desired cell iscontemplated as within the scope of the present invention. The site ofadministration and cells will be selected by one of ordinary skill inthe art based upon an understanding of the particular disorder beingtreated. In addition, the dosage, dosage frequency, and length of courseof treatment, can be determined and optimized by one of ordinary skillin the art depending upon the particular degenerative disorder beingtreated. The particular mode of administration can also be readilyselected by one of ordinary skill in the art and can include, forexample, oral, intravenous, subcutaneous, intramuscular, etc., with therequirement that the therapeutic agent cross the cell membrane.Principles of pharmaceutical dosage and drug delivery are known and aredescribed, for example, in Ansel, H. C. and Popovich, N. G.,Pharmaceutical Dosage Forms and Drug Delivery Systems, 5th Edition, Lea& Febiger, Publisher, Philadelphia, Pa. (1990). It is possible, forexample, to utilize liposomes to specifically deliver the agents of theinvention. Such liposomes can be produced so that they containadditional bioactive compounds and the like such as drugs,radioisotopes, antibodies, lectins and toxins, which would act at thetarget site.

Suitable agents for use according to the invention include GD domainpeptides and mimetics, fragments, functional equivalents and/or hybridsor mutants thereof, as well as vectors containing cDNA encoding any ofthe foregoing. Agents can be administered alone or in combination withand/or concurrently with other suitable drugs and/or courses of therapy.

The agents of the present invention are suitable for the treatment ofdegenerative disorders, including disorders characterized byinappropriate cell proliferation or inappropriate cell death or in somecases, both. Inappropriate cell proliferation will include thestatistically significant increase in cell number as compared to theproliferation of that particular cell type in the normal population.Also included are disorders whereby a cell is present and/or persists inan inappropriate location, e.g., the presence of fibroblasts in lungtissue after acute lung injury. For example, such cells include cancercells which exhibit the properties of invasion and metastasis and arehighly anaplastic. Such cells include but are not limited to, cancercells including, for example, tumor cells. Inappropriate cell death willinclude a statistically significant decrease in cell number as comparedto the presence of that particular cell type in the normal population.Such underrepresentation may be due to a particular degenerativedisorder, includinG, for example, AIDS (HIV), which results in theinappropriate death of T-cells, and autoimmune diseases which arecharacterized by inappropriate cell death. Autoimmune diseases aredisorders caused by an immune response directed against self antigens.Such diseases are characterized by the presence of circulatingautoantibodies or cell-mediated immunity against autoantigens inconjunction with inflammatory lesions caused by immunologicallycompetent cells or immune complexes in tissues containing theautoantigens. Such diseases include systemic lupus erythematosus (SLE),rheumatoid arthritis.

Standard reference works setting forth the heneral principles ofimmunology include Stites, D. P., and Terr, A. I., Basic and ClinicalImmunology, 7th Ed., Appleton & Lange, Publisher, Norwalk, Conn. (1991);and Abbas, A. K., et al., Cellular and Molecular Immunology, W. B.Saunders Co., Publisher, Philadelphia, Pa. (1991).

The GD domain peptides, mimetics, agents and the like disclosed herein,as well as vectors comprising nucleotide sequences encoding them ortheir corresponding antisense sequences, and hosts comprising suchvectors, may be used in the manufacture of medicaments for the treatmentof diseases.

Cells and non-human transgenic animals having one or more functionallyimpaired alleles encoding a protein comprising the GD domain may begenerated using homologous targeting constructs from genomic clones ofproteins comprising the GD domain. Methods for the production ofhomologous targeting constructs are known and described, for example, inBradley, et al., Bio/Technology 10: 534 (1992); and Koh, et al., Science256: 1210 (1992). For example, "knock-out" mice may be generated whichare homozygous or heterozygous for an inactivated allele of a proteincomprising the GD domain byuse of homologous targeting. Such mice areuseful as research subjects for the investigation of disease and forother uses. Methods of producing chimeric targeted mice are known andare described, for example, in Robertson, E. J., Ed., Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, IRL Press, Washington,D.C. (1987), which also describes the manipulation of embryonic stemcells. In addition, transgenes for expressing polypeptides comprisingthe GD domain at high levels or under the control of selectedtranscription control sequences may be constructed using the cDNA orgenomic gene of a protein comprising the GD domain. Transgenes soconstructed can be introduced into cells and transgenic non-humananimals by known methods. Such transgenic cells and transgenic non-humananimals may be used as screens for agents which modulate apoptosis.

The invention may be appreciated in certain aspects with reference tothe following examples, offered by way of illustration, not by way oflimitation.

EXAMPLES A. Methods 1. Plasmids and DNA Manipulations.

All recombinant DNA procedures were performed by standard methods.Deletions in the bak cDNA were introduced by PCR mutagenesis, andtruncated Bak species were constructedby PCR (White, B. A., Ed., "PCRProtocols: Current Methods and Applications," in, Methods in MolecularBiology, Humana Press, Totowa, Conn. (1993). The mutations wereconfirmed by DNA sequence analysis. All Bak derivatives were tagged atthe amino-terminus with influenza virus hemagglutinin epitope, andexpressed from the CMV enhancer promoter present in pcDNA-1/Amp, pRcCMV,and pcDNA-3 (Invitrogen, Inc.).

2. Transient Transfection Assay.

The transient transfection assay procedure is similar to that previouslydescribed for detecting apoptosis induced by IL-1β-converting enzyme(Miura et al., Cell 75: 653-660 (1993); Kumar et al., Genes Dev. 8:1613-1626 (1994); Wang et al., Cell 78: 739-750 (1994). One day prior totransfection, Rat-1 cells were plated in 24 well dishes at 3.5×10⁴cells/well. The following day, the cells were transfected with a markerplasmid encoding β-galactosidase (0.16 μg), in combination with anexpression plasmid encoding Bak (0.42 82 g), by the Lipofectamineprocedure (Gibco/BRL). At 24 hours post transfection, cells were fixedand stained with X-Gal to detect β-galactosidase expression in cellsthat received plasmid DNA (Miura et al., supra). The number of bluecells was counted by microscopic examination and scored as either live(flat blue cells) or dead (round blue cells). The cell killing activityof Bak in this assay is manifested by a large reduction in the number ofblue cells obtained relative to co-transfection of the β-gal plasmidwith a control expression vector (i.e., with no bak cDNA insert).

The interpretation that loss of blue cells reflects the cell killingfunction of Bak is supported by a variety of observations:

1. Rat-1 cells are rapidly killed by enforced Bak expression in stabletransfection assays;

2. Control expression plasmids harboring the bak cDNA in the anti-senseorientation, or various unrelated cDNAs, do not eliminate β-gal positivecells. In addition, certain Bak mutants (i.e., ΔGD) have greatlydiminished capacity to eliminate blue cells in this assay;

3. IL-β-converting enzyme, previously shown to induce apoptosis in Rat-1cells (Miura et al., supra; Kumar et al., supra; Wang et al., supra),also eliminates blue cells in this assay when expressed from the samevector;

4. The number of blue cells can be partially restored by co-transfectionof Bak with Bcl-x_(L). Thus, Bak expressing cells can be rescued to somedegree by the anti-apoptotic function of Bcl-x_(L), and Bak expressionper se does not eliminate β-galactosidase activity.

3. Detection of Protein/Protein Interactions in Vitro.

GST and GST-Bcl-x_(L) were expressed in E. coli and purified by affinitychromatography using glutathione-agarose (Smith and Johnson, Gene 67:31-40 (1988)). ³⁵ S-Methionine-labeled proteins were expressed in vitrousing a coupled transcription/translation system in rabbit reticulocytelysates as described by the supplier (Promega). ³⁵ S-met-labeledproteins were precleared by mixing with 20 ml BSA-washed GSH-agarosebeads (50% slurry) at 4° C. for 1 hour in 0.1 ml 10 mM Hepes buffer, pH7.2 containing 0 25% NP-40, 142.5 mM NaCl, 5 mM MgCl₂, and 1 mM EGTA(NP-40 lysis buffer). The beads were removed by centrifugation and thesupernatants were incubated with GST or GST-Bcl-x_(L) (finalconcentration 1 mM) at 4° C. for 1 hour. The GST fusion proteins and anyinteracting proteins were recovered by incubation for 1 hour with anadditional 20 ml of GSH-agarose beads. The beads were washed twice withNP-40 lysis buffer followed by two washes with NP-40 lysis bufferwithout NP-40. Proteins were eluted from the beads by incubation inSDS-PAGE sample buffer at 100° C. for 5 min and loaded onto 4-20%SDS-polyacrylamide gels. Following electrophoresis, gels were fixed andincubated in a fluorography enhancing solution (Amplify; Amersham). Thegels were dried and subjected to autoradiography at -70° C.

4. Detection of Protein/Protein interactions in Transfected Cells.

COS cells were grown in Dulbecco's modified Eagle's medium (LifeTechnologies, Inc.) supplemented with 10% bovine calf serum, 2%L-glutamine and 1% pen/strep (Life Technologies, Inc.). Cells wereseeded at 2.0×10⁵ cells/ 35 mm well and transfected with expressionplasmids 24 hours later using Lipofectamine as described by the supplier(Life Technologies, Inc.). Bak (and Bak mutants) was expressed as afusion protein with the HA epitope tag at its amino terminus. Bcl-x_(L)was also expressed with an amino terminal epitope tag (Flag; Kodak). At24 hours post-transfection, cells were washed with phosphate bufferedsaline and lysed in NP-40 Lysis buffer also containing 1 mM PMSF, 1 mMpepstatin, and 1 mg/ml leupeptin. The lysates were incubated withanti-HA antibody (12CA5, Boehringer Mannheim) for 1 hour and with 20 mlBSA-washed Protein A-agarose beads (50% slurry) for an additional hour.The beads were washed twice with NP-40 lysis buffer followed by twowashes with NP-40 lysis buffer without NP-40. Proteins were eluted fromthe beads by incubation in SDS-PAGE sample buffer at 100° C. for 5 minand loaded onto 4-20% SDS-polyacrylamide gels. Followingelectrophoresis, proteins were transferred to Immobilon-P membranes(Millipore) and the membranes were blocked by incubation for 1 hour witha 1% milk solution in PBS. Primary antibody (1 mg/ml 12CA5, BoehringerMannheim; 1:500 DAKO-bcl-2, 124, DAKO; 10 mg/ml Anti-FLAG M2, Kodak) wasincubated with the membranes for 1 hour, followed by secondary antibody(0.8 mg/ml HRP-conjugated goat anti-mouse IgG; Jackson Laboratory) foran additional 1 hour. Detection was by enhanced chemiluminesence (ECL;Amersham) as described by the supplier using X-OMAT AR film (Kodak).

B. Results 1. Detection of the Cell Death Function of Bak in MultipleCell Lines.

Enforced bak expression induces apoptosis in stable Rat-1 cell linestransfected with an inducible bak expression plasmid. In order to morerapidly assess the cell killing function of a large number of bakmutants, a transient transfection assay was employed. Rat-1 cells weretransfected with a marker plasmid encoding β-galactosidase, incombination with an expression plasmid encoding Bak, or various controlplasmids. Cell killing activity of Bak in this assay was manifested by alarge reduction in the number of blue (β-gal expressing) cells obtainedrelative to co-transfection of the β-gal plasmid with a controlexpression vector (FIG. 1). The elimination of blue cells indicated thattransfected cells were killed by bak prior to expressing detectablelevels of β-galactosidase.

Bak cell killing activity was assessed in several additional cell lines.To determine whether Bak requires wild-type p53 to induce apoptosis, atransient transfection experiment was performed in transformedfibroblasts derived from a p53-/- "knockout" mouse. These cells lackfunctional p53 and are greatly impaired in their ability to undergoapoptosis in response to g-irradiation and DNA-damaging chemotherapeuticdrugs (Lowe et al., Cell 74: 957-967 (1993); Lowe et al., Nature 362:847-849 (1993)). Co-transfection of Bak with β-gal greatly reduced thenumber of blue cells (FIG. 1) indicating that Bak does not requirewild-type p53 to exert its cell killing function. Similarly, transienttransfection experiments performed in the Hela (cervical carcinoma) andBT549 (breast carcinoma) cell lines demonstrated that Bak can kill humantumor cells in this context (FIG. 1) indicating that its activity is notrestricted to rodent fibroblasts.

2. Identification of Bak Domains Required for Cell Killing Function.

A mutational analysis of Bak was undertaken in order to identify regionsof the molecule that are necessary and/or sufficient to induceapoptosis. A series of deletion mutations spanning the entire Bakprotein was introduced by PCR mutagenesis and each mutant was tested forcell killing activity in a Rat-1 cell transient transfection assay. Thisanalysis revealed that much of the Bak molecule is dispensable for itscell death function detected by this assay (FIG. 2). Surprisingly, thenon-essential regions of the Bak protein include the two domains in thecarboxyl terminal half of the protein that show the highest degree ofhomology to other Bcl-2 family members (Bcl-2 homology domains I andII).

Deletion of the carboxyl-terminal hydrophobic stretch of amino acids(residues 191-211) partially diminished, but did not eliminate, the cellkilling function of Bak (mutant ΔC). This hydrophobic "tail" likelyserves as a membrane anchor sequence in Bak. Indeed, immunofluorescencestudies of ΔC in transiently transfected COS cells showed that theintracellular distribution of the AC mutant is altered (diffusecytoplasmic) relative to the wild type Bak, which appears largelymitochondrial. The carboxyl terminal hydrophobic tail is not requiredfor the cell killing function of Bak, but may contribute indirectly, byensuring proper sub-cellular localization of the protein.

A segment of the Bak protein encompassed by the ΔGD deletion (residues82-94) is absolutely required for cell death function since this mutantis devoid of cell killing activity in the transient transfection assay.Specifically, co-transfection of β-gal with Bak AGD yielded as many, ormore, blue cells relative to co-transfection of β-gal with the controlvector plasmid. Deletion of adjoining residues (amino acids 67-81)immediately N-terminal to this domain reduced, but did not eliminate,cell death activity (Bak mutant ΔPS). All other deletion mutants tested(with the exception of ΔC, discussed above) were unaltered in theircapacity to kill cells. Taken together, these results indicate that aco-linear segment (termed the "GD domain") defined by deletion mutantsΔGD and ΔPS (residues 67-94) is uniquely required for Bak cell killingfunction detected in the transient assay.

To determine if the GD domain is sufficient for cell killing function,two truncated Bak protein derivatives, PEM and QVG, corresponding toamino acids 58-103 and 73-123, respectively, were tested for activity inthe transient transfection assay. QVG significantly reduced the numberof blue cells when co-transfected with β-gal, indicating that itretained some capacity to kill Rat-1 cells. While the reduction in bluecell number was diminished relative to full length Bak, both PEM and QVGlack the carboxyl-terminal membrane anchor and, by analogy to the Bak ΔCmutant, would likely not exhibit full cell killing function due toaltered sub-cellular localization. Indeed, QVG was similar to the Bak ΔCmutant with respect to its activity. In an effort to improve the cellkilling capacity of the truncated Bak species, the hydrophobic tailelement (amino acids 187-211) was fused to the C-termini of both PEM andQVG (PEM+C and QVG+C, respectively). In each case, attachment of theputative membrane anchor improved the ability of the truncated Bakmutants to eliminate blue cells in the transfection assay, and resultedin activity comparable to wild-type Bak (FIG. 2). Thus, these resultsindicate that a protein domain shared by both PEM and QVG (residues73-103) is sufficient for the cell killing function of Bak.

3. Identification of Bak Domains That Mediate the Interaction withBcl-x_(L)

Physical interaction with other Bcl-2 family members, such as Bcl-x_(L),may be essential for Bak to exert its cell death function or mayregulate Bak activity. Therefore, domains within Bak were examined todetermine which are necessary and/or sufficient for its Bcl-x_(L)binding activity. The interaction of Bak with Bcl-x_(L) was measuredboth by an in vitro protein binding assay andby co-immunoprecipitationfrom transfected cells. In vitro translated ³⁵ S labeled Bak binds to apurified, bacterially expressed GST-Bcl-x_(L) fusion protein, and thespecificity of this in vitro interaction was demonstratedby the failureof Bak to bind to purified GST alone (FIG. 3A). A specific Bak/Bcl-x_(L)interaction could also be detected by co-transfecting epitope taggedforms of Bak and Bcl-x_(L) into COS cells. Bak was immunoprecipitatedfrom transfected cell lysates and associated Bcl-x_(L) was detected byWestern blot analysis of co-precipitated proteins (FIG. 3B). Bcl-x_(L)was not detected in immunoprecipitates in the absence of co-expressedBak, demonstrating that binding is specific.

The Bak deletion mutants described above were tested for their Bcl-x_(L)binding capacity, both in vitro and in transfected COS cells, and theresults are summarized in FIG. 4. Deletion of residues 82-94 (ΔGDmutant) completely eliminated the ability of Bak to interact withBcl-x_(L). Interaction with Bcl-x_(L) was also diminished by deletion ofadjoining amino acids 67-81 (ΔPS Bak mutant). All other deletion mutantstested, encompassing the entire Bak open reading frame, retained theability to bind Bcl-x_(L) in these assays. These results identify Baksequences encompassed by the ΔGD and ΔPS mutants (maximally, amino acids67-94) as uniquely important in mediating the interaction withBcl-x_(L). The same Bak region, the GD domain, was required for the cellkilling function of Bak.

To determine whether the Bak region defined by deletion analysis issufficient for protein binding function, two small truncated Bak species(PEM and QVG), encompassing amino acids 58-103 and 73-123 respectively,were tested for their ability to interact with Bcl-x_(L). Both PEM andQVG bound Bcl-x_(L), indicating that the region shared by both of thesetruncated Bak species (amino acids 73-103) was sufficient for mediatingthe interaction with Bcl-x_(L). Together with the analysis of thedeletion mutants and truncated species described above, these resultsdemonstrate that Bak amino acid sequences spanning residues 73-103 areboth necessary and sufficient for interaction with Bcl-xL. As describedabove, this region is also implicated in the cell killing function ofBak, indicating that protein binding function may linked to cell killingfunction.

4. Functionally Significant Sequence Elements Resembling the GD DomainAre Present in Bax and Bip1la.

The mutational analysis of Bak described herein demonstrates that the GDdomain is uniquely involved in both the cell killing and Bcl-x_(L)binding activities of Bak. Two other Bcl-2 interacting proteins, Bax andBip1a, have functional properties that resemble those of Bak. Both Baxand Bip1a eliminate blue cells when co-transfected with β-gal in Rat-1cells, indicating that they also induce apoptosis in this context. Baxand Bip1a also interact specifically with Bcl-x_(L), both in vitro andin transfected COS cells. These functional similarities prompted theexamination of whether any structural features are shared by the threeproteins that contribute to their similar biological functions.Specifically, in light of the analysis presented above, Bax and Bip1awere examined to determine whether they contain sequences that resemblethe Bak GD domain and are also important for their biologicalactivities.

Bax shows extensive homology to Bcl-2 family members (including Bak),with the highest degree of sequence homology centered around BH1 and BH2(Oltvai et al., Cell 74: 609-619 (1993)). A stretch of amino acids(59-73) N-terminal to BH1 in Bax bears homology to sequences (residues74-88) within the GD domain of Bak (FIG. 5). In contrast to Bax, theprimary sequence of Bip1a does not resemble the known Bcl-2 relatives,and lacks sequences homologous to BH1 and BH2 that are characteristic ofthe Bcl-2 family. However, Bip1a contains a region (amino acids 57-71)that is homologous to the same element within the GD domain in Bak andBax (FIG. 5).

GD domain elements within Bax and Bip1a were evaluated to determinewhether they are also critical to the cell killing and protein bindingfunctions of these proteins. Small deletions that removed the conservedGD domain motifs were introduced into Bax and Bip1a, and the mutantswere then analyzed for their ability to kill Rat-1 cells and bind toBcl-x_(L). This analysis revealed that, like Bak ΔGD, the Bax ΔGD andBip1a ΔGD mutants are impaired in their ability to eliminate blue cellswhen co-transfected with β-Gal in Rat-1 cells (FIG. 6). In addition,both mutants no longer have the capacity to interact with Bcl-x_(L)(FIG. 6). Thus, function of the GD domain element is conserved in Bak,Bax and Bip1a, and is critical to the biological activities of all threeproteins.

5. The GD Domain is Sufficient for Homo- and Heterodimer Formation.

In order to assess whether the GD domain mediates other protein/proteininteractions which could be relevant to its biological activity, aportion of Bak (PEM) encompassing the GD domain (residues 58-103) wasfused to GST, to create GST-PEM. In vitro translated, ³⁵ S labeledBcl-x_(L), Bak, Bax and Bip1a were incubated with either GST alone, orGST-PEM bacterially-expressed fusion protein. Interactions of the GDdomain with Bak and Bax were measured essentially as described hereinfor Bak binding to Bcl-x_(L). Complexes were captured withglutathione-agarose beads, washed, and bound proteins detected bypolyacrylamide gel electrophoresis and autoradiography.

The results of this experiment are shown in FIG. 6. Bcl-x_(L), Bak, andBax all interact specifically with GST-PEM, but not with GST alone.These results demonstrate that the Bak GD domain is sufficient to bindto Bak (homodimerization), Bax (heterodimerization with a differentkiller protein) and Bcl-x_(L) (heterodimerization with a survivalprotein). Thus, the GD domain is capable of mediating interactions notonly with Bcl-x_(L), but also Bak and Bax. It does not interact withBip1a.

All publications mentioned in this specification are herein incorporatedby reference, to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

It will be understood that the invention is capable of furthermodifications and this application is intended to cover any variations,uses, or adoptions of the invention including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains, and is intended to be limited onlyby the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 34                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acid                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlyAspAspIleAsnArgArgTyrAspSerGluPheGln                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acid                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ProSerSerThrMetGlyGlnValGlyArgGlnLeuAlaIleIleGly                              51015                                                                         AspAspIleAsnArgArgTyrAspSerGluPheGln                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acid                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GlnValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg                              51015                                                                         TyrAspSerGluPheGlnThrMetLeuGlnHisLeuGlnProThr                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acid                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       LeuSerGluCysLeuLysArgIleGlyAspGluLeuAspSerAsn                                 51015                                                                         (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       LeuLysArgIleGlyAspGluLeuAsp                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       GlnAspAlaSerThrLysLysLeuSerGluCysLeuLysArgIleGly                              51015                                                                         AspGluLeuAspSerAsnMetGluLeuGln                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       LeuAlaLeuArgLeuAlaCysIleGlyAspGluMetAspValSer                                 51015                                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       IleGlyAspGluMet                                                               5                                                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysMetGluGlySerAspAlaLeuAlaLeuAspLeuAlaCysIleGly                              51015                                                                         AspGluMetAspValSerLeuArgAlaProArgLeu                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      ValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg                                 51015                                                                         (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AlaAlaProAlaAspProGluMetValThrLeuProLeuVal                                    510                                                                           (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      LeuGluCysMetGluGlySerAspAlaLeuAlaLeuArgLeuAlaCys                              51015                                                                         IleGlyAspGluMetAspValSerLeuArgAlaProArgLeuAlaGln                              202530                                                                        LeuSerGluVal                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 34 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      ValProGlnAspAlaSerThrLysLysLeuSerGluCysLeuLysArg                              51015                                                                         IleGlyAspGluLeuAspSerAsnMetGluLeuGlnArgMetIleAla                              202530                                                                        AlaVal                                                                        (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base pairs                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      LeuGlnProSerSerThrMetGlyGlnValGlyArgGlnLeuAlaIle                              51015                                                                         IleGlyAspAspIleAsnArgArgTyrAspSerGluPheGlnThrMet                              202530                                                                        LeuGlnHisLeu                                                                  35                                                                            (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 93 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CAGGTGGGACGGCAGCTCGCCATCATCGGGGACGACATCAACCGACGCTATGACTCAGAG60                TTCCAGACCATGTTGCAGCACCTGCAGCCCACG93                                           (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GlnValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg                              51015                                                                         TyrAspSerGluPheGlnThrMetLeuGlnHisLeuGlnProThr                                 202530                                                                        (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      CCTAGCAGCACCATGGGGCAGGTGGGACGGCAGCTCGCCATCATCGGGGACGACATCAAC60                CGACGCTATGACTCAGAGTTCCAG84                                                    (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      ProSerSerThrMetGlyGlnValGlyArgGlnLeuAlaIleIleGly                              51015                                                                         AspAspIleAsnArgArgTyrAspSerGluPheGln                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      GTGGGACGGCAGCTCGCCATCATCGGGGACGACATCAACCGACGC45                               (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      ValGlyArgGlnLeuAlaIleIleGlyAspAspIleAsnArgArg                                 51015                                                                         (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      GGGGACGACATCAACCGACGCTATGACTCAGAGTTCCAG39                                     (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      GlyAspAspIleAsnArgArgTyrAspSerGluPheGln                                       510                                                                           (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 78 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CAGGATGCGTCCACCAAGAAGCTGAGCGAGTGTCTCAAGCGCATCGGGGACGAACTGGAC60                AGTAACATGGAGCTGCAG78                                                          (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 26 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      GlnAspAlaSerThrLysLysLeuSerGluCysLeuLysArgIleGly                              51015                                                                         AspGluLeuAspSerAsnMetGluLeuGln                                                2025                                                                          (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      CTGAGCGAGTGTCTCAAGCGCATCGGGGACGAACTGGACAGTAAC45                               (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      LeuSerGluCysLeuLysArgIleGlyAspGluLeuAspSerAsn                                 51015                                                                         (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      CTCAAGCGCATCGGGGACGAACTGGAC27                                                 (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      LeuLysArgIleGlyAspGluLeuAsp                                                   5                                                                             (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TGCATGGAGGGCAGTGACGCATTGGCCCTGCGGCTGGCCTGCATCGGGGACGAGATGGAC60                GTGAGCCTGAGGGCCCCGCGCCTG84                                                    (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      CysMetGluGlySerAspAlaLeuAlaLeuArgLeuAlaCysIleGly                              51015                                                                         AspGluMetAspValSerLeuArgAlaProArgLeu                                          2025                                                                          (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      TTGGCCCTGCGGCTGGCCTGCATCGGGGACGAGATGGACGTGAGC45                               (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino acids                                                    (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      LeuAlaLeuArgLeuAlaCysIleGlyAspGluMetAspValSer                                 51015                                                                         (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      ATCGGGGACGAGATG15                                                             (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      IleGlyAspGluMet                                                               5                                                                             __________________________________________________________________________

What is claimed is:
 1. An isolated and purified peptide having an aminoacid sequence selected from the group consisting of amino acid sequencesset forth as SEQ ID NOS:1-10.
 2. A composition comprising the isolatedand purified peptide according to claim 1 and a carrier.